Preparation and Detection Method of Fluorescence Immunoassay Chromatographic Detection Card for Lead and Cadmium in Tobacco

Abstract

The present invention belongs to the technical field of immunoassay, and discloses a preparation method and a detection method of a fluorescence immunoassay chromatography test strip for lead and cadmium in tobacco. The method includes: preparing a horseradish peroxidase solution; preparing an immobilized enzyme-MOFs complex; preparing a labeled antigen; drawing a T line at one end of the NC membrane close to the sample loading end, and drawing a C line at the other end of the NC membrane far from the sample loading end; mixing the immobilized enzyme-MOFs complex, the labeled antigen and the sample diluent according to a volume ratio of 2:3:5, and uniformly spraying the mixture on the glass fiber membrane as a conjugate pad, pasting a blotting paper at the other end of the NC membrane, covering the PVC upper cover, and pressing tightly to complete the assembly of the test strip. The present invention effectively reduces the interference of short-lived background fluorescence in tobacco samples, greatly improves the detection accuracy, can simultaneously detect two heavy metal elements of lead and cadmium, and can save time and detection costs.

Description

Technical Field

[0001] The present invention relates to the technical field of immunoassay, and more specifically, to a preparation method and a detection method for a fluorescence immunoassay chromatography test card for lead and cadmium in tobacco. Background Art

[0002] With the development of the tobacco industry and the continuous improvement of people's attention to health, the problem of heavy metal pollution in tobacco has received increasing attention. During the growth process of tobacco, it is easy to absorb heavy metal elements from the soil, water source, and atmospheric environment. Among them, lead and cadmium are the two most common and harmful heavy metals.

[0003] Chinese patent application with publication number CN116047061A discloses a time-resolved fluorescence immunoassay chromatography test strip for detecting heavy metal cadmium ions, its preparation method and application, including: preparing an europium time-resolved fluorescence microsphere-heavy metal cadmium monoclonal antibody immune complex; preparing a conjugate pad sprayed with the europium time-resolved fluorescence microsphere-heavy metal cadmium monoclonal antibody immune complex; coating heavy metal cadmium antigen on the T line and goat anti-mouse secondary antibody on the C line, and after drying, pasting it together with absorbent paper, sample pad, and the conjugate pad to assemble a time-resolved fluorescence immunoassay chromatography test strip for detecting heavy metal cadmium ions. The preparation method of the time-resolved fluorescence immunoassay chromatography test strip for detecting heavy metal cadmium ions constructed by this invention is simple. By establishing the relationship between the concentration of the substance to be measured and the fluorescence intensity, it can realize the detection of cadmium ions in food, and has a fast detection speed and high sensitivity, and is suitable for on-site detection in situations such as farmland, supermarkets, and families. Specifically, it can be applied to the detection of cadmium ions in tea, and has important practical significance and application value in the quality control of tea.

[0004] Although the above method can meet most scenarios, through research and practical application of the above method and the existing technology, it is found that the above method and the existing technology have at least the following partial defects:

[0005] The time-resolved fluorescence immunoassay chromatography test strip mainly detects cadmium ions and cannot provide detection information for lead elements; it mainly relies on the natural reaction process of the cadmium monoclonal antibody immune complex and the antigen on the test strip, without additional complexes to assist in accelerating the reaction or enhancing the signal; resulting in a slower reaction speed, weaker signal generation, and relatively lower sensitivity when detecting low-concentration cadmium ions, and unable to meet the precise detection requirements for trace cadmium pollution in tobacco.

[0006] In view of this, the present invention proposes a preparation method and a detection method for a fluorescence immunoassay chromatography test card for lead and cadmium in tobacco to solve the above problems. Summary of the Invention

[0007] To overcome the above-mentioned defects of the prior art, the present invention provides the following technical solutions: A preparation method for a fluorescence immunoassay chromatography test strip for lead and cadmium in tobacco, comprising the following steps:

[0008] Take 5-20 g of MOFs powder and add it to a horseradish peroxidase solution with a concentration of 0.5-2 mg / mL. Stir in a 4°C environment and incubate for 2-4 h. Collect the immobilized enzyme-MOFs complex by centrifugal separation, and wash it repeatedly with a PBS buffer solution with a pH of 7.0-7.4 and a concentration of 0.01-0.1 mol / L. Resuspend the immobilized enzyme-MOFs complex in a PBS buffer solution with a pH of 7.0-7.4 and a concentration of 0.01-0.1 mol / L, and store it for later use in a 4°C environment;

[0009] Couple lead antigen with time-resolved fluorescence microspheres to prepare a first labeled antigen; couple cadmium antigen with time-resolved fluorescence microspheres to prepare a second labeled antigen; mix the first labeled antigen and the second labeled antigen in a volume ratio of 1:1 to obtain a labeled antigen;

[0010] Paste an NC membrane with different pore sizes and surface chemical properties in the middle position of the PVC bottom plate. Draw a T line at one end of the NC membrane with different pore sizes and surface chemical properties close to the sample loading end. Use a micro-sampler to evenly spot the lead and cadmium specific antibodies on the T line at intervals of 0.5-1.5 μL and 3-5 mm, and air dry; draw a C line at the other end of the NC membrane with different pore sizes and surface chemical properties far from the sample loading end, and evenly spot the goat anti-mouse IgG antibody at intervals of 0.5-1.5 μL and 3-5 mm, and air dry for 30-60 min;

[0011] Mix the immobilized enzyme-MOFs complex, the labeled antigen and the sample diluent in a volume ratio of 2:3:5, and evenly spray them on the glass fiber membrane as a conjugate pad. Paste the absorbent paper at the other end of the NC membrane with different pore sizes and surface chemical properties, cover the PVC upper cover, and press tightly to complete the assembly of the test strip.

[0012] Furthermore, the preparation method of the MOFs powder includes;

[0013] Dissolve dry zinc nitrate in N,N-dimethylformamide, place it on a magnetic stirrer and stir until completely dissolved to prepare a first mixed solution with a concentration of 0.1-0.3 mol / L;

[0014] Dissolve recrystallized phthalic acid in N,N-dimethylformamide, place it on a magnetic stirrer and stir until completely dissolved to prepare a first composite solution with a concentration of 0.05-0.15 mol / L;

[0015] Transfer the first mixed solution and the first composite solution into a stainless-steel reactor lined with polytetrafluoroethylene according to a molar ratio of 1:1 - 2, and control the filling degree of the reactor to be 60% - 80%; seal the reactor and place it in an oven at a temperature of 120 - 150 °C for reaction for 12 - 36 h to form MOFs crystals;

[0016] Turn off the oven and wait for the reactor to cool naturally to room temperature; filter the reaction solution in the reactor through filter paper, collect the solid product on the filter paper, preliminarily separate the MOFs crystals and the reaction solvent, wash the collected solid product with DMF 3 - 5 times repeatedly; then transfer the washed MOFs crystals to a Soxhlet extractor, use absolute ethanol as the extractant, and extract for 24 - 48 h; place the extracted MOFs crystals in a vacuum drying oven and dry at 80 - 100 °C for 10 - 20 h to obtain MOFs powder.

[0017] Furthermore, the method for preparing the labeled antigen includes:

[0018] Step 1: Disperse the blank microspheres in a terbium chloride solution with a concentration of 0.01 - 0.1 mol / L at 20 - 60 °C, control the magnetic stirring speed at 100 - 500 rpm for magnetic stirring, after reacting for 12 - 48 h, centrifuge at 5000 - 10000 rpm for 5 - 15 min to discard the supernatant, obtain the microsphere precipitate, and wash the microsphere precipitate with an ethanol solution with a volume fraction of 70% - 95% to obtain time-resolved fluorescence microspheres;

[0019] Step 2: Activate the time-resolved fluorescence microspheres to obtain activated time-resolved fluorescence microspheres, and modify the activated time-resolved fluorescence microspheres based on polyethylene glycol derivatives to obtain modified time-resolved fluorescence microspheres;

[0020] Step 3: Couple the lead antigen solution and the modified time-resolved fluorescence microspheres according to a molar ratio of 5 - 15:1 to obtain a first coupling solution; couple the cadmium antigen solution and the modified time-resolved fluorescence microspheres according to a molar ratio of 5 - 15:1 to obtain a second coupling solution;

[0021] Step 4: Add BSA blocking solution to the first coupling solution and the second coupling solution respectively to block the vacant sites on the surface of the modified time-resolved fluorescence microspheres to obtain the first labeled antigen and the second labeled antigen;

[0022] Step 5: Mix the first labeled antigen and the second labeled antigen according to a volume ratio of 1:1 to obtain the labeled antigen.

[0023] Furthermore, the method for obtaining the modified time-resolved fluorescence microspheres includes:

[0024] Step 2.1: Suspend time-resolved fluorescence microspheres with a volume of 1 - 10 mL and a concentration of 5 mg / mL in a PBS buffer solution with a volume of 10 - 50 mL and a concentration of 0.01 - 0.1 mol / L. The PBS buffer solution contains EDC and NHS, and the concentrations of both EDC and NHS are 1 - 10 mM. Stir and react at a stirring speed of 200 - 400 rpm at room temperature for 30 - 60 min, then obtain a centrifugate through centrifugation at a speed of 5000 - 10000 rpm for 5 - 15 min, and remove the supernatant of the centrifugate. Wash the precipitate with the PBS buffer solution 2 - 3 times; perform centrifugation at a speed of 5000 - 10000 rpm for 5 - 15 min after each washing, and obtain the precipitate from the last centrifugation as the activated time-resolved fluorescence microspheres;

[0025] Step 2.2: Resuspend the activated time-resolved fluorescence microspheres in a PBS buffer solution with a volume of 10 - 50 mL and a concentration of 0.01 - 0.1 mol / L. The PBS buffer solution contains NHS-PEG with a concentration of 1 - 10 mM, and stir at a stirring speed of 100 - 300 rpm at room temperature for 2 - 24 h to obtain a modification solution;

[0026] Step 2.3: Perform precipitation treatment on the modification solution through centrifugation at 5000 - 10000 rpm for 5 - 15 min, remove the supernatant to obtain a precipitate, wash the precipitate 3 - 5 times with a PBS buffer solution with a volume of 10 - 50 mL and a concentration of 0.01 - 0.1 mol / L, and perform centrifugation at 5000 - 10000 rpm for 5 - 15 min after each washing to obtain the precipitate from the last centrifugation as the modified time-resolved fluorescence microspheres.

[0027] Furthermore, the method for obtaining the NC membranes with different pore sizes and surface chemical properties includes:

[0028] Slowly add 5 - 15 g of nitrocellulose with a purity greater than 95% and a molecular weight of 80000 - 150000 Da to a mixed solvent with a mass fraction of 10% - 15% and a volume of 50 - 150 mL, stir and dissolve at a stirring speed of 200 - 400 rpm at room temperature until a uniform and transparent mixed solution is formed; wherein, the mixed solvent is a mixed solvent of acetone and DMF mixed in a volume ratio of 3 - 5:1;

[0029] Add 0.3 - 0.9 g of PEG2000, 0.5 - 1.5 g of PEG6000, and 0.2 - 0.6 g of PEG20000 to the mixed solution respectively, and continue to stir at a stirring speed of 200 - 400 rpm for 2 - 4 h to obtain a second mixed solution;

[0030] Slowly add 0.15 - 0.45 g of 3 - aminopropyltriethoxysilane to the second mixed solution, and stir and react at a stirring speed of 200 - 400 rpm for 1 - 2 h to obtain a third mixed solution;

[0031] Add 0.015 - 0.045 g of sodium dodecyl sulfate to the third mixed solution, and stir for 30 - 60 min to obtain a prepared solution;

[0032] Pour the prepared solution onto a clean glass plate placed horizontally, and use a scraper to uniformly scrape the film at a speed of 5 - 10 cm / s and a gap of 100 - 200 μm to obtain a liquid film;

[0033] Immediately transfer the liquid film together with the glass plate to an environmental chamber at a temperature of 25 - 35 °C and a humidity of 40% - 60%, and place it for 12 - 24 h until the liquid film is completely dry;

[0034] After the liquid film is dry, peel it off from the glass plate, and rinse it 2 - 3 times with deionized water, 3 - 5 min each time, to obtain a thin film;

[0035] Soak the thin film in absolute ethanol for 1 - 2 h, then take it out and place it in a fume hood to dry;

[0036] Dry the dried thin film in a vacuum oven at 40 - 50 °C for 4 - 6 h to obtain the first NC membrane;

[0037] Soak the first NC membrane in absolute ethanol for 10 - 30 min to obtain the pretreated first NC membrane;

[0038] Slowly add 10 - 50 mL of 3 - aminopropyltriethoxysilane with a concentration of 0.5 - 1.5% to absolute ethanol, and stir at a stirring speed of 100 - 300 rpm for 10 - 30 min at room temperature to obtain an APTES solution;

[0039] Soak the pretreated first NC membrane in the APTES solution at 20 - 40 °C for 1 - 6 h, then take it out to obtain the second NC membrane;

[0040] Take out the second NC membrane from the APTES solution and rinse it 3 - 5 times with absolute ethanol to obtain the third NC membrane;

[0041] Soak the third NC membrane in PBS buffer at room temperature for 10 - 30 min, then rinse it 2 - 3 times with deionized water to obtain the fourth NC membrane;

[0042] Dry the fourth NC membrane in a vacuum oven at 40 - 60 °C for 2 - 6 h; and cut the dried product to obtain NC membranes with different pore sizes and surface chemical properties.

[0043] Detection method for a fluorescence immunochromatographic test strip for lead and cadmium in tobacco, comprising the following steps:

[0044] Take 5 - 10 g of tobacco sample, cut it into pieces with a particle size of 1 - 3 mm, put it into a container containing 10 - 15 mL of aminotrimethylphosphonic acid with a concentration of 0.02 - 0.08 mol / L, and connect the container to the inlet of the micro-nano extraction device;

[0045] Turn on the ultrasonic generator, set the ultrasonic frequency to 20 - 50 kHz and the power to 100 - 300 W, and extract for 5 - 10 minutes;

[0046] After the extraction, use a hydrochloric acid solution with a concentration of 0.01 - 0.1 mol / L as the eluent to elute the lead and cadmium ions adsorbed on the extraction material, and collect the eluent as the sample to be tested for subsequent immunoassay;

[0047] Drop 100 - 200 μL of the sample to be tested into the sample well of the test strip prepared by the preparation method for a fluorescence immunochromatographic test strip for lead and cadmium in tobacco, and incubate for 10 - 15 min;

[0048] Insert the test strip into the supporting time-resolved fluorescence immunoassay analyzer, read the signal intensity values of the T line and the C line, and process the signal intensity values to obtain the standard concentrations of lead and cadmium in tobacco.

[0049] Furthermore, the method for processing the signal intensity values to obtain the standard concentrations of lead and cadmium in tobacco includes:

[0050] Preset wavelength and , where is the emission wavelength of the labeled antigen; is the reference wavelength;

[0051] Before inserting the test strip and without adding the sample to be tested, start the background monitoring program of the detection instrument; the instrument collects the fluorescence signal intensity at the positions of the T line and the C line at the preset frequency, continuously collects for 10 - 20 s, and obtains a set of background signal intensity data, denoted as , where represents the emission wavelength of the selected antigen or the reference wavelength , represents the number of acquisitions;

[0052] Calculate the average value and the standard deviation of the background signal intensity at the two wavelengths respectively; where is the total number of acquisitions, and take as the initial background subtraction value at wavelength and As the wavelength of the initial background subtraction value, and set the confidence interval to , where is a constant.

[0053] Furthermore, the standard sample extract with known lead and cadmium concentrations prepared in advance is dropped onto the test card, inserted into the time-resolved fluorescence immunoassay analyzer, and the real-time signal intensity values of the T line and the C line at the wavelengths and are obtained and denoted as , , and , where is the real-time signal intensity of the T line at the wavelength ; is the real-time signal intensity of the T line at the wavelength ; is the real-time signal intensity of the C line at the wavelength ; is the real-time signal intensity of the C line at the wavelength ; is the th standard sample; at the same time, the background signal intensity is collected;

[0054] Calculate the signal intensity ratios and of the T line at the wavelengths , and the signal intensity ratios and of the C line at the wavelengths ;

[0055] For each standard sample, calculate its corresponding lead and cadmium concentration ratio , where and are the concentrations of lead and cadmium in the th standard sample respectively;

[0056] Use the least squares method to fit and establish the linear relationship between the T line correction coefficient and the C line correction coefficient and the concentration ratio :

[0057] For the T line: , solve the coefficients and the intercept by the least squares method;

[0058] For the C line: , similarly solve the coefficients and the intercept ;

[0059] Further, collect the total signal intensities of the T-line and C-line at two wavelengths at a preset time interval. 、 、 and , where is the total signal intensity of the T-line at wavelength ; is the total signal intensity of the T-line at wavelength ; is the total signal intensity of the C-line at wavelength ; is the total signal intensity of the C-line at wavelength ; At the same time, collect the background signal intensity , where , corresponding to the background signal intensity at wavelength and the background signal intensity at wavelength respectively;

[0060] If is within the confidence interval, then use as the background subtraction value and calculate the net signal intensity:

[0061] ;

[0062] ;

[0063] ;

[0064] ;

[0065] where is the net signal intensity of the T-line at wavelength ; is the net signal intensity of the T-line at wavelength ; is the net signal intensity of the C-line at wavelength ; is the net signal intensity of the C-line at wavelength ;

[0066] If exceeds the confidence interval, then recalculate the background average , where is the number of times of the re-collected background signal, and use as the new background subtraction value to update the net signal intensity;

[0067] Calculate the effective signal intensity of the compensated T-line and the effective signal intensity of the C-line :

[0068] ;

[0069] ;

[0070] Among them, is the estimated ratio of the lead and cadmium concentrations in the real-time detection sample;

[0071] Using the standard curve established in advance with standard samples, combined with the effective signal intensity of the compensated T line , the concentrations of lead and cadmium in the sample to be tested are calculated by interpolation or regression equation.

[0072] Furthermore, the methods for calculating the concentrations of lead and cadmium in the sample to be tested by interpolation or regression equation include:

[0073] Collect the coordinates of two adjacent data points of the effective signal intensity of the compensated T line on the standard curve ; and , among which, the effective signal intensity of the compensated T line is located and ; the concentrations of lead and cadmium in the sample to be tested are calculated by the following formula:

[0074] ,

[0075] Among them, is the concentration of lead and cadmium in the sample to be tested.

[0076] The technical effects and advantages of the preparation and detection method of the fluorescence immunoassay chromatography detection card for lead and cadmium in tobacco of the present invention:

[0077] In the preparation process of the present invention, by combining horseradish peroxidase with MOFs powder to form an immobilized enzyme-MOFs complex, it helps to enhance the stability and efficiency of the immune response; combining rare earth elements with blank microspheres under specific conditions to form microspheres with a long fluorescence decay time can effectively avoid the interference of short-lived background fluorescence in tobacco samples. By setting an appropriate delay time to collect signals, the detection signal-to-noise ratio and accuracy are greatly improved, and the reliability of lead and cadmium detection in tobacco is enhanced. Activating and modifying the time-resolved fluorescence microspheres with polyethylene glycol derivatives makes them have better water solubility and biocompatibility, reducing non-specific adsorption, thereby improving the accuracy and sensitivity of detection. By preparing NC membranes with different pore sizes and surface chemical properties and applying them to the detection card, the migration speed of substances in tobacco samples and the impurity retention effect can be effectively regulated, ensuring that the antigen-antibody reaction is sufficient and not interfered, and the antibody and antigen can be firmly fixed, enhancing the repeatability and stability of the detection card. During the detection process, a unique sample pretreatment method combined with a precise signal intensity processing algorithm can effectively extract lead and cadmium ions in tobacco, accurately deduct the background signal, compensate the effective signal, and accurately obtain the concentration of lead and cadmium in tobacco through a standard curve and various calculation methods, realizing the efficient, accurate, and rapid detection of lead and cadmium in tobacco, providing strong technical support for tobacco heavy metal pollution monitoring. Brief Description of the Drawings

[0078] Figure 1 It is a schematic diagram of the fluorescence immunochromatographic detection card for lead and cadmium in tobacco in Example 1 of the present invention;

[0079] Figure 2 It is a schematic cross-sectional structure diagram of the fluorescence immunochromatographic detection card for lead and cadmium in tobacco in Example 1 of the present invention;

[0080] Figure 3 It is a schematic diagram of the standard curve of concentration and fluorescence intensity in Example 2 of the present invention;

[0081] Figure 4 It is a schematic diagram showing the change of the fluorescence value of the T line with the addition amount of horseradish peroxidase powder in Example 3 of the present invention;

[0082] Figure 5 It is a schematic diagram showing the change of the fluorescence value of the T line with the addition amount of SM-PEG24 in Example 4 of the present invention;

[0083] Figure 6 It is a schematic diagram of the detection result of the detection card in Example 5 of the present invention;

[0084] Figure 7 It is a schematic diagram showing the change of the fluorescence value of the T line with the coupling molar ratio in Example 6 of the present invention;

[0085] Figure 8Schematic diagram of the fluorescence value of the T line varying with the concentration of aminotrimethylphosphonic acid in Example 7 of the present invention;

[0086] Figure 9 Schematic diagram of the false positive rate detection result of the test card in Example 8 of the present invention;

[0087] Figure 10 Schematic diagram of the false positive rate detection result of the test card in Example 9 of the present invention;

[0088] Figure 11 Schematic diagram of the test result of the test card in Example 10 of the present invention.

[0089] Meanings of the reference numerals: 1, quality control line; 2, test line; 3, sample hole; 4, sample pad; 5, conjugate pad; 6, NC membrane; 7, PVC upper cover; 8, absorbent paper; 9, PVC bottom plate. Detailed implementation manners

[0090] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0091] Example 1

[0092] Please refer to Figure 1 - Figure 2 as shown, the preparation method of the fluorescence immunochromatographic test card for lead and cadmium in tobacco in this embodiment includes the following steps:

[0093] Weigh 25 - 100 mg of horseradish peroxidase powder and slowly add it to 50 mL of pre-prepared PBS buffer solution, where the pH of the PBS buffer solution is 7.0 - 7.4 and the concentration is 0.01 - 0.1 mol / L. Gently stir with a glass rod in a 4°C environment until evenly mixed to obtain a horseradish peroxidase solution with a concentration of 0.5 - 2 mg / mL;

[0094] Take 5 - 20 g of MOFs powder and add it to the horseradish peroxidase solution. Gently stir with a glass rod in a 4°C environment and incubate for 2 h - 4 h. Collect the immobilized enzyme - MOFs complex by centrifugal separation, and wash it repeatedly with PBS buffer solution with a pH of 7.0 - 7.4 and a concentration of 0.01 - 0.1 mol / L. Resuspend the immobilized enzyme - MOFs complex in PBS buffer solution with a pH of 7.0 - 7.4 and a concentration of 0.01 - 0.1 mol / L, and store it for standby in a 4°C environment;

[0095] The preparation method of the MOFs powder includes;

[0096] Dissolve dry zinc nitrate in N,N-dimethylformamide, place it on a magnetic stirrer and stir until completely dissolved to prepare a first mixed solution with a concentration of 0.1 - 0.3 mol / L;

[0097] Dissolve recrystallized phthalic acid in N,N-dimethylformamide, place it on a magnetic stirrer and stir until completely dissolved to prepare a first composite solution with a concentration of 0.05 - 0.15 mol / L;

[0098] Transfer the first mixed solution and the first composite solution to a stainless steel reaction kettle with a PTFE lining in a ratio of 1:1 - 2 in terms of molar ratio, and control the filling degree of the reaction kettle to be 60% - 80%; Seal the reaction kettle and place it in an oven at a temperature of 120 - 150 °C for reaction for 12 - 36 h to form MOFs crystals;

[0099] Turn off the oven and wait for the reaction kettle to cool naturally to room temperature; Filter the reaction solution in the reaction kettle through filter paper, collect the solid product on the filter paper, preliminarily separate the MOFs crystals and the reaction solvent, and wash the collected solid product with DMF 3 - 5 times repeatedly; Then transfer the washed MOFs crystals to a Soxhlet extractor, use absolute ethanol as the extractant, and extract for 24 - 48 h; Place the extracted MOFs crystals in a vacuum drying oven and dry at 80 - 100 °C for 10 - 20 h to obtain MOFs powder.

[0100] Couple lead antigen with time-resolved fluorescence microspheres to prepare a first labeled antigen; Couple cadmium antigen with time-resolved fluorescence microspheres to prepare a second labeled antigen; Mix the first labeled antigen and the second labeled antigen in a volume ratio of 1:1 to obtain a labeled antigen;

[0101] The preparation method of the labeled antigen is as follows:

[0102] Under the condition of 20 - 60 °C, disperse the blank microspheres in a terbium chloride solution with a concentration of 0.01 mol / L - 0.1 mol / L, control the magnetic stirring speed at 100 - 500 rpm for magnetic stirring, after reacting for 12 - 48 h, centrifuge at 5000 - 10000 rpm for 5 - 15 min and discard the supernatant to obtain the microsphere precipitate. Wash the microsphere precipitate with an ethanol solution with a volume fraction of 70% - 95% to obtain time-resolved fluorescence microspheres; when the blank microspheres are dispersed in the terbium chloride solution, through physical adsorption, chemical bonding or electrostatic interaction, etc., the terbium chloride solution can be adsorbed or bound on the surface of the microspheres to form a coating doped with rare earth elements, thus obtaining fluorescence microspheres doped with rare earth elements; microspheres with long-lived fluorescence characteristics can be selected accordingly. Their fluorescence decay time is significantly extended compared with ordinary fluorescent substances, which can effectively avoid the interference of short-lived background fluorescence in tobacco samples. During detection, an appropriate delay time can be set to collect signals. After the short-lived background fluorescence fades, then detect the fluorescence emitted by the microspheres, greatly improving the signal-to-noise ratio.

[0103] Perform activation treatment on the time-resolved fluorescence microspheres to obtain activated time-resolved fluorescence microspheres, and modify the activated time-resolved fluorescence microspheres based on polyethylene glycol derivatives to obtain modified time-resolved fluorescence microspheres;

[0104] The method for obtaining the modified time-resolved fluorescence microspheres includes:

[0105] Suspend time-resolved fluorescence microspheres with a volume of 1 - 10 mL and a concentration of 5 mg / mL in a PBS buffer solution with a volume of 10 - 50 mL and a concentration of 0.01 - 0.1 mol / L. The PBS buffer solution contains EDC and NHS, and the concentrations of EDC and NHS are both 1 - 10 mM; control the stirring speed at 200 - 400 rpm at room temperature and stir for 30 - 60 min, then obtain the centrifugate through centrifugation at a speed of 5000 - 10000 rpm for 5 - 15 min, and remove the supernatant of the centrifugate; wash the precipitate with the PBS buffer solution 2 - 3 times; perform centrifugation at a speed of 5000 - 10000 rpm for 5 - 15 min after each washing, and obtain the precipitate from the last centrifugation as the activated time-resolved fluorescence microspheres;

[0106] Resuspend the activated time-resolved fluorescence microspheres in a PBS buffer solution with a volume of 10 - 50 mL and a concentration of 0.01 - 0.1 mol / L. The PBS buffer solution contains NHS-PEG, and the concentration of NHS-PEG is 1 - 10 mM. Control the stirring speed at 100 - 300 rpm at room temperature and stir for 2 - 24 h to obtain the modification solution;

[0107] The modified solution was precipitated by centrifugation at 5000 - 10000 rpm for 5 - 15 min, and the supernatant was removed to obtain the precipitate. The precipitate was washed 3 - 5 times with PBS buffer having a volume of 10 - 50 mL and a concentration of 0.01 - 0.1 mol / L. After each washing, centrifugation was carried out at 5000 - 10000 rpm for 5 - 15 min. The precipitate obtained from the last centrifugation was used as the modified time-resolved fluorescence microspheres. Modifying the surface of the time-resolved fluorescence microspheres can make the time-resolved fluorescence microspheres have better water solubility and biocompatibility, and reduce non-specific adsorption.

[0108] The lead antigen was added to the modified time-resolved fluorescence microsphere solution according to a molar ratio of 5 - 15:1 for mixing, and the centrifuge tube was placed in a constant temperature water bath at 25 - 30 °C and continuously stirred and reacted for 1 - 2 h;

[0109] 1 - 3% BSA blocking solution was added to block the vacant sites on the surface of the modified time-resolved fluorescence microspheres to obtain the first labeled antigen;

[0110] The second labeled antigen was prepared according to the same steps as those for preparing the first labeled antigen.

[0111] The first labeled antigen and the second labeled antigen were mixed according to a volume ratio of 1:1 to obtain a labeled antigen with a concentration of 0.1 - 0.5 μg / mL.

[0112] The NC membrane 6 with different pore sizes and surface chemical properties was pasted at the middle position of the PVC bottom plate 9. A T line was drawn at one end of the NC membrane 6 with different pore sizes and surface chemical properties close to the sample loading end. The lead-cadmium specific antibody was evenly spotted on the T line with a micro-sampler at intervals of 0.5 - 1.5 μL and 3 - 5 mm, and then dried. A C line was drawn at the other end of the NC membrane 6 with different pore sizes and surface chemical properties far from the sample loading end. The goat anti-mouse IgG antibody was evenly spotted at intervals of 0.5 - 1.5 μL and 3 - 5 mm, and then dried for 30 - 60 min;

[0113] The method for obtaining the NC membrane 6 with different pore sizes and surface chemical properties includes:

[0114] 5 - 15 g of nitrocellulose with a purity greater than 95% and a molecular weight of 80000 - 150000 Da was slowly added to a mixed solvent with a mass fraction of 10% - 15% and a volume of 50 - 150 mL, and stirred and dissolved at a stirring speed of 200 - 400 rpm at room temperature until a uniform and transparent mixed solution was formed; wherein, the mixed solvent is a mixed solvent of acetone and DMF mixed according to a volume ratio of 3 - 5:1;

[0115] Add 0.3 - 0.9 g of PEG2000, 0.5 - 1.5 g of PEG6000, and 0.2 - 0.6 g of PEG20000 to the mixed solution respectively, and continue stirring at a stirring speed of 200 - 400 rpm for 2 - 4 h to obtain a second mixed solution;

[0116] Slowly add 0.15 - 0.45 g of 3 - aminopropyltriethoxysilane to the second mixed solution, and stir and react at a stirring speed of 200 - 400 rpm for 1 - 2 h to obtain a third mixed solution;

[0117] Add 0.015 - 0.045 g of sodium dodecyl sulfate to the third mixed solution and stir for 30 - 60 min to obtain a prepared solution;

[0118] Slowly pour the prepared solution onto a clean glass plate placed horizontally, and use a scraper to uniformly scrape the film at a speed of 5 - 10 cm / s and a gap of 100 - 200 μm to obtain a liquid film;

[0119] Immediately transfer the liquid film together with the glass plate to an environmental chamber at a temperature of 25 - 35 °C and a humidity of 40% - 60% and place it for 12 - 24 h until the liquid film is completely dry;

[0120] After the liquid film is dried, peel it off from the glass plate, and rinse it 2 - 3 times with deionized water, 3 - 5 min each time, to obtain a thin film;

[0121] Soak the thin film in absolute ethanol for 1 - 2 h and then take it out and place it in a fume hood to dry;

[0122] Dry the dried thin film in a vacuum oven at 40 - 50 °C for 4 - 6 h to obtain a first NC membrane;

[0123] Soak the first NC membrane in absolute ethanol for 10 - 30 min to obtain a pretreated first NC membrane;

[0124] Slowly add 10 - 50 mL of 3 - aminopropyltriethoxysilane with a concentration of 0.5 - 1.5% to absolute ethanol, and stir at a stirring speed of 100 - 300 rpm for 10 - 30 min at room temperature to obtain an APTES solution;

[0125] Soak the pretreated first NC membrane in the APTES solution at 20 - 40 °C for 1 - 6 h and then take it out to obtain a second NC membrane;

[0126] Take out the second NC membrane from the APTES solution and rinse it 3 - 5 times with absolute ethanol to obtain a third NC membrane;

[0127] After soaking the third NC membrane in PBS buffer at room temperature for 10 - 30 min, rinse it 2 - 3 times with deionized water to obtain the fourth NC membrane;

[0128] Dry the fourth NC membrane in a vacuum oven at 40 - 60 °C for 2 - 6 h; and cut the dried product to obtain NC membranes 6 with different pore sizes and surface chemical properties. Obtaining chromatography membranes with different pore sizes and surface chemical properties can enable the chromatography membrane to prevent impurity particles in tobacco samples from passing through while ensuring the effective binding and migration of antibodies and antigens, reducing false positive results. And introducing a special functional group, amino group, on the surface of the chromatography membrane can immobilize antibodies or other capture reagents, improving the immobilization efficiency and stability of antibodies, thereby enhancing the repeatability and accuracy of the test strip.

[0129] The method for obtaining lead and cadmium specific antibodies includes:

[0130] Primary immunization: Mix the first labeled antigen and Freund's complete adjuvant in a volume ratio of 1:1, place it in an ice bath, and use an emulsifier to fully emulsify until a first water-in-oil emulsion is formed; Select 6 - 8 week-old BALB / c mice and inject the first water-in-oil emulsion with a dose of 100 - 200 μL for the mice; Inject the second water-in-oil emulsion prepared from the second labeled antigen and Freund's complete adjuvant with a dose of 100 - 200 μL for the mice in the same way;

[0131] Perform the first booster immunization 2 - 3 weeks after the primary immunization, and the operation is the same as the primary immunization process.

[0132] Subcutaneously inject the corresponding group of mice, and the injection dose for each mouse can be appropriately increased to 200 - 300 μL; Then repeat the booster immunization step every 2 - 3 weeks, generally perform 3 - 4 booster immunizations to enhance the immune response of the mice and prompt them to produce antibodies with high affinity and high specificity.

[0133] Take the spleen cells of the immunized mice and fuse them with myeloma cells, and obtain a cell line that stably secretes lead and cadmium specific monoclonal antibodies through hybridoma screening technology. Prepare lead specific antibodies and cadmium specific antibodies using the ascites method or cell culture method, and purify them for standby.

[0134] Mix the immobilized enzyme-MOFs complex, labeled antigen, and sample diluent in a volume ratio of 2:3:5, and then evenly spray the mixture onto a glass fiber membrane to serve as the conjugate pad 5. Paste one end of the NC membrane 6 with different pore sizes and surface chemical properties, on which the control line 1 and the test line 2, that is, the C line and the T line, are spot-coated, to the conjugate pad 5. Paste the other end of the conjugate pad 5 to the sample pad 4. Paste the absorbent paper 8 to the other end of the NC membrane 6 with different pore sizes and surface chemical properties. After drying, paste it above the PVC bottom plate 9, cover the PVC top cover 7, and press it tightly to complete the assembly of the test card. The pasting method of the test card can be specifically referred to Figure 2 . The appearance of the test card can be referred to Figure 1 . There is a test window above the test card. In the test window, the control line 1, that is, the C line, is above, and the test line 2, that is, the T line, is below. Below the test window is the sample hole 3 for dropping the test sample.

[0135] Example 2

[0136] The detection method of the fluorescence immunochromatographic test card for lead and cadmium in tobacco in this example includes the following steps:

[0137] Take 5-10 g of tobacco samples, cut them into pieces with a particle size of 1-3 mm, put them into a container containing 10-15 mL of aminotrimethylphosphonic acid with a concentration of 0.02-0.08 mol / L, and connect the container to the inlet of the micro-nano extraction device;

[0138] Turn on the ultrasonic generator, set the ultrasonic frequency to 20-50 kHz and the power to 100-300 W, and extract for 5-10 minutes;

[0139] After the extraction is completed, use a hydrochloric acid solution with a concentration of 0.01-0.1 mol / L as the eluent to elute the lead and cadmium ions adsorbed on the extraction material, and collect the eluate as the test sample for subsequent immunoassay;

[0140] Drop 100-200 μL of the test sample onto the sample hole of the test card prepared by the preparation method of the fluorescence immunochromatographic test card for lead and cadmium in tobacco in Example 1, and incubate for 10-15 min;

[0141] Insert the test card into the supporting time-resolved fluorescence immunoassay analyzer, read the signal intensity values of the T line and the C line, and process the signal intensity values to obtain the standard concentration of lead and cadmium in tobacco.

[0142] A series of test samples with different concentrations can also be prepared and operated according to the above detection steps. Using the concentration of the test sample standard solution as the abscissa and the ratio of the signal intensity of the T line to the C line as the ordinate, a standard curve is plotted; specifically, it can be referred to Figure 3 , and the standard curve equation is obtained through linear regression analysis.

[0143] A method for obtaining the standard concentration of lead and cadmium in tobacco by processing the signal intensity values includes:

[0144] Preset wavelength And , where Is the emission wavelength of the labeled antigen; Is the reference wavelength;

[0145] Before inserting the test card and without adding the sample to be tested, start the background monitoring program of the detection instrument; the instrument collects the fluorescence signal intensity at the positions of the T line and the C line at a preset frequency, continuously collects for 10 - 20 s, and obtains a set of background signal intensity data, denoted as , where Represents the emission wavelength of the selected antigen Or the reference wavelength Represents the number of acquisitions;

[0146] Calculate the average value And the standard deviation Of the background signal intensity at the two wavelengths respectively; where Is the total number of acquisitions, and take As the initial background subtraction value at wavelength And As the initial background subtraction value at wavelength , and set the confidence interval to , where Is a constant;

[0147] Drop the standard sample extract with a known lead and cadmium concentration prepared in advance onto the test card, insert it into the time-resolved fluorescence immunoassay analyzer, and obtain the real-time signal intensity values of the T line and the C line at wavelengths And , denoted as , , And , where Is the real-time signal intensity of the T line at wavelength ; Is the real-time signal intensity of the T line at wavelength ; Is the real-time signal intensity of the C line at wavelength ; Is the real-time signal intensity of the C line at wavelength ; Is the th standard sample; at the same time, collect the background signal intensity at this time;

[0148] Calculate the T line at wavelengths And The signal intensity ratio under , and the signal intensity ratios of the C line at wavelengths and ; ;

[0149] For each standard sample, calculate its corresponding lead-cadmium concentration ratio , where and are the concentrations of lead and cadmium in the th standard sample, respectively;

[0150] Use the least squares method to fit and establish the linear relationship between the T-line correction coefficient and the C-line correction coefficient and the concentration ratio :

[0151] For the T line: , solve for the coefficient and the intercept using the least squares method;

[0152] For the C line: , similarly solve for the coefficient and the intercept ;

[0153] Collect the total signal intensities of the T line and the C line at two wavelengths at preset time intervals , , and , where is the total signal intensity of the T line at wavelength ; is the total signal intensity of the T line at wavelength ; is the total signal intensity of the C line at wavelength ; is the total signal intensity of the C line at wavelength ; At the same time, collect the background signal intensity , where corresponds to the background signal intensity at wavelength and the background signal intensity at wavelength respectively;

[0154] If is within the confidence interval, then use as the background subtraction value to calculate the net signal intensity:

[0155] ;

[0156] ;

[0157] ;

[0158] ;

[0159] Wherein, is the net signal intensity of the T line at wavelength ; is the net signal intensity of the T line at wavelength ; is the net signal intensity of the C line at wavelength ; is the net signal intensity of the C line at wavelength ;

[0160] If exceeds the confidence interval, recalculate the background average value , wherein, is the number of times of the re - collected background signal, and use as the new background subtraction value to update the net signal intensity;

[0161] Calculate the effective signal intensity of the compensated T line and the effective signal intensity of the C line:

[0162] ;

[0163] ;

[0164] Wherein, is the estimated ratio of the lead and cadmium concentrations in the real - time detection sample;

[0165] Using the standard curve established in advance through standard samples, combined with the effective signal intensity of the compensated T line, calculate the concentrations of lead and cadmium in the sample to be measured by interpolation or regression equation;

[0166] The method for calculating the concentrations of lead and cadmium in the sample to be measured by interpolation or regression equation includes:

[0167] Collect the coordinates of two adjacent data points of the effective signal intensity and of the compensated T line on the standard curve, wherein, the effective signal intensity of the compensated T line is between and ; Calculate the concentrations of lead and cadmium in the sample to be measured by the following formula:

[0168]

[0169] Among them, is the concentration of lead and cadmium in the sample to be tested.

[0170] Example 3

[0171] Optimization of enzyme loading in the immobilized enzyme-MOFs composite:

[0172] During the preparation of the immobilized enzyme-MOFs composite, the concentration of horseradish peroxidase solution affects the amount of HRP enzyme immobilized per gram of MOFs powder, that is, the enzyme loading; too high or too low enzyme loading will affect the accuracy of later detection of heavy metals lead and cadmium; therefore, the addition amount of horseradish peroxidase powder is explored. The preparation method of the immobilized enzyme-MOFs composite is the same as that in Example 1, except that: the addition amounts of horseradish peroxidase powder are 10, 25, 50, 75, 100, 115 and 150 μL respectively, and the corresponding fluorescence values of the T line are compared. The relationship between the addition amount of horseradish peroxidase powder and the fluorescence value is as Figure 4 shown. When the addition amount of horseradish peroxidase powder in the immobilized enzyme-MOFs composite is 75 mg, the effect is the best.

[0173] Example 4

[0174] Optimization of activator concentration:

[0175] During the time-resolved fluorescence microsphere labeling antibody process, first, the carboxyl groups on the surface of the fluorescence microspheres need to be activated. The quality of the activated microspheres is crucial for the subsequent labeling process. The dosage of the activator determines the activation degree of the carboxyl groups on the microsphere surface. After screening out the better activation conditions, the addition amount of the activator is explored. The preparation method of the carbendazim antibody-fluorescence microsphere label is the same as that in Example 1, except that: SM-PEG24 is added to the diluted microspheres according to the molar ratio of microspheres to SM-PEG24 of 1:1, 1:3, 1:5, 1:7, 1:10, 1:13 respectively, and the corresponding fluorescence values of the T line are compared. The relationship between the addition amount of SM-PEG24 and the fluorescence value is as Figure 5 shown. When 6 μL of SM-PEG24 is added to the labeled antigen, the effect is the best.

[0176] Example 5

[0177] Optimization of the modification of time-resolved fluorescence microspheres:

[0178] During the process of labeling antibodies with time-resolved fluorescent microspheres, modifying the surface of the time-resolved fluorescent microspheres can make them have better water solubility and biocompatibility, reduce non-specific adsorption, protect the fluorescence performance of quantum dots, and enhance stability in the immunochromatography system. The preparation method of the carbendazim antibody-fluorescent microsphere label is the same as that in Example 1, and the detection method is the same as that in Example 2, with the only difference being that the time-resolved fluorescent microspheres are respectively treated with only activation, activation + modification, and only modification. After a positive result appears in the detection, it is still left standing for 30 min, and samples containing heavy metals lead and cadmium are detected with the same batch of test strips. The results under ultraviolet light irradiation are as Figure 6 shown. All three groups showed positive results. However, in the control group with only activation, after a positive result appeared in the detection and it was left standing for a period of time, the color development became lighter; in the experimental group with activation + modification, after a positive result appeared in the detection and it was left standing for a period of time, the color development remained unchanged; in the control group with only modification, the positive result shown during the detection had a lighter color development, and after it was left standing for a period of time, the color development also became lighter. This shows that the modification of the time-resolved fluorescent microspheres according to the present invention can protect the fluorescence performance of quantum dots and enhance stability in the immunochromatography system.

[0179] Example 6

[0180] Optimization of the coupling molar ratio of antigen to time-resolved fluorescent microspheres:

[0181] During the process of labeling microspheres with antigen, the molar ratio of the lead and cadmium antigen solution to the microsphere solution is crucial for the subsequent labeling process. The molar ratio can ensure that there is enough antigen to react with the active groups on the surface of the microspheres. If the antigen ratio is too low, the active groups on the surface of the microspheres cannot fully react, resulting in low coupling efficiency and insufficient activity of the labeled antigen; if the antigen ratio is too high, too many antigen molecules bind to the surface of the microspheres, which may cause steric hindrance. This will hinder the specific binding of antigen and antibody and affect the accuracy of detection. After screening out the relatively optimal antigen labeling conditions, the coupling molar ratio was explored. The preparation method of the labeled antigen is the same as that in Example 1, with the only difference being that the coupling molar ratios are 1:1, 3:1, 5:1, 7:1, 10:1, 15:1, and 18:1, and the corresponding fluorescence values of the T line were compared. The results of the relationship between the coupling molar ratio and the fluorescence value are as Figure 7 shown. The best effect is achieved when the coupling molar ratio in the labeled antigen is 10:1.

[0182] Example 7

[0183] Optimization of the extract concentration:

[0184] During the process of processing tobacco samples, the concentration of the extraction solution affects the extraction efficiency of lead and cadmium ions, and further affects the efficiency of detecting the heavy metal lead and cadmium content in tobacco samples in the later stage. Therefore, the concentration of the extraction solution is further explored. Aminotrimethylphosphonic acid is selected as the extraction solution, and the method for detecting heavy metals lead and cadmium in tobacco samples is the same as that in Example 2, except that: the concentrations of aminotrimethylphosphonic acid are 0.01 mol / L, 0.02 mol / L, 0.04 mol / L, 0.06 mol / L, 0.08 mol / L, and 0.1 mol / L; the corresponding fluorescence values of the T line are compared. The relationship between the concentration of aminotrimethylphosphonic acid and the fluorescence value is as Figure 8 shown, and the best effect is obtained when the concentration of aminotrimethylphosphonic acid is 0.06 mol / L.

[0185] Example 8

[0186] Optimization of the pore size distribution of the NC membrane:

[0187] During the detection process of the chromatographic test card, the pore size distribution of the NC membrane affects the detection results of tobacco samples. A suitable pore size distribution helps the effective migration of lead and cadmium ions and other substances such as labeled antigens in tobacco samples on the NC membrane. If the pore size is too large, the sample solution may quickly pass through the NC membrane, resulting in too short a binding time of antigens and antibodies on the membrane, affecting the sensitivity and accuracy of detection. For example, when the pore size exceeds a certain threshold, it may cause some unreacted labeled antigens to directly pass through the T line and the C line, resulting in deviation of the detection signal. The preparation method of the NC membrane is the same as that in Example 1, except that the NC membrane is prepared only from PEG2000, only from PEG6000, only from PEG20000, from a mixture of PEG2000 and PEG6000, from a mixture of PEG6000 and PEG20000, from a mixture of PEG2000 and PEG20000, and from a mixture of PEG2000, PEG6000, and PEG20000. The detection method is the same as that in Example 2; the tobacco samples are detected the same number of times, such as 30 times, and are sequentially labeled as group 1, group 2, group 3, group 4, group 5, group 6, and group 7; the false positive rate of all detection results is statistically calculated, that is, the percentage of the recognition results in which tobacco samples without lead and cadmium are identified as positive in the total number of detections, specifically as Figure 9 shown. It can be seen from the figure that group 7, that is, the NC membrane prepared from a mixture of PEG2000, PEG6000, and PEG20000, has the best detection effect.

[0188] Example 9

[0189] Optimization of the chemical properties of the NC membrane:

[0190] During the detection process of the chromatographic test strip, the chemical properties of the NC membrane determine its ability to immobilize antibodies and antigens. If functional groups are introduced into the NC membrane, the functional groups can form stable chemical bonds or strong physical adsorption with antibody or antigen molecules, making the immobilization of antibodies and antigens on the NC membrane more firm and uniform. The preparation method of the NC membrane is the same as that of Example 1, except that the NC membrane is prepared by soaking in APTES solution and without soaking in APTES solution respectively; the detection method is the same as that of Example 2; the tobacco samples are detected the same number of times, such as 100 times, and are labeled as Group 1 and Group 2 in sequence. All the detection results are statistically analyzed, specifically as Figure 10 shown. It can be seen from the figure that the detection effect of Group 2, that is, the NC membrane prepared by soaking in APTES solution, is the best.

[0191] Example 10

[0192] Specificity verification:

[0193] Select several common heavy metal elements, such as chromium, nickel, lead and cadmium, for specificity tests. Prepare heavy metal element standards at a concentration of 2000 μg / mL, and use the same batch of test strips to conduct interference tests on the above heavy metal element standards. The standard without heavy metal elements is used as a negative control. The test results are shown in Table 1. The detections of chromium and nickel show negative results. The T values of the detections of lead and cadmium are significantly lower than those of chromium and nickel, and the C values are comparable. The T / C value can represent the ratio of the fluorescence intensities of the test line and the quality control line, and is used for the quantification of the corresponding antigen in the sample. Under ultraviolet light irradiation, the results are as Figure 11 shown. The standards containing lead and cadmium show positive results, and the rest are negative, indicating that the present invention has good specificity for the heavy metal elements lead and cadmium and has no cross-reaction with other heavy metal elements.

[0194] Table 1 Specific detection data of different heavy metal elements

[0195] Heavy metal elements Average T value Average T value T / C value Blank control 15364 969 15.86 Chromium 15343 975 15.74 Nickel 14572 986 14.78 Lead 2453 996 2.46 Cadmium 2448 994 2.46

[0196] It can be seen from Table 1 that the test strip provided by the present invention has high specificity and accurate measurement, can effectively avoid the interference of other substances, ensure that the detection results accurately reflect the true situation of lead and cadmium in the sample; and is easy to operate, can quickly conduct preliminary detections of the lead and cadmium contents of a large number of tobacco samples, timely discover the problem of heavy metal over-standard, improve work efficiency, and can also ensure the quality of tobacco raw materials.

[0197] In summary, the present invention labels antigens on the surface of fluorescent microspheres, and amplifies the binding signal of antigens and antibodies by detecting the fluorescence value of the microspheres. This method has significant advantages in detecting low-concentration antigens or antibodies; compared with the traditional method of directly detecting antigen-antibody binding, it can detect target substances at lower concentrations and can detect antigens or antibodies at the nanogram or picogram level. The time-resolved fluorescence immunochromatographic test strip established thereby for quantitatively detecting heavy metals lead and cadmium in tobacco samples has a higher detection accuracy and better effect; moreover, the present invention selects aminotrimethylphosphonic acid with a concentration of 0.06 mol / L as an extraction solution for pretreatment of tobacco samples, and combines an ultrasonic extraction method, which can efficiently extract heavy metal ions from tobacco, provide accurate samples for subsequent detection, help to more precisely evaluate the heavy metal content in tobacco, and ensure the quality and safety of tobacco products.

[0198] As described above, the above is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present invention can easily think of changes or substitutions, which should all be covered within the protection scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.

[0199] Finally: The above is only a preferred embodiment of the present invention and is not used to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention shall all be included within the protection scope of the present invention.

Claims

1. Preparation method of fluorescence immuno-chromatographic detection card for lead and cadmium in tobacco, characterized in that It includes the following steps: Take 5 - 20 g of MOFs powder and add it to a horseradish peroxidase solution with a concentration of 0.5 - 2 mg / mL. Stir and incubate for 2 - 4 h at 4°C. Collect the immobilized enzyme - MOFs complex by centrifugation, wash it repeatedly with a PBS buffer solution with a pH of 7.0 - 7.4 and a concentration of 0.01 - 0.1 mol / L, resuspend the immobilized enzyme - MOFs complex in a PBS buffer solution with a pH of 7.0 - 7.4 and a concentration of 0.01 - 0.1 mol / L, and store it at 4°C for later use; Couple lead antigen with time - resolved fluorescence microspheres to prepare the first labeled antigen; couple cadmium antigen with time - resolved fluorescence microspheres to prepare the second labeled antigen; mix the first labeled antigen and the second labeled antigen in a volume ratio of 1:1 to obtain the labeled antigen; Paste NC membranes with different pore sizes and surface chemical properties in the middle position of the PVC bottom plate. Draw a T - line at one end of the NC membrane with different pore sizes and surface chemical properties close to the sample loading end. Use a micro - sampler to evenly spot the lead - cadmium specific antibody obtained by mixing lead - specific monoclonal antibody and cadmium - specific monoclonal antibody in a volume ratio of 1:1 on the T - line at intervals of 0.5 - 1.5 μL and 3 - 5 mm, and air - dry; draw a C - line at the end of the NC membrane with different pore sizes and surface chemical properties far from the sample loading end, evenly spot goat anti - mouse IgG antibody at intervals of 0.5 - 1.5 μL and 3 - 5 mm, and air - dry for 30 - 60 min; Mix the immobilized enzyme - MOFs complex, the labeled antigen and the sample diluent in a volume ratio of 2:3:5, and then evenly spray them on the glass fiber membrane as the conjugate pad. Paste the absorbent paper at the other end of the NC membrane with different pore sizes and surface chemical properties, cover the PVC upper cover, and press tightly to complete the assembly of the test card; The method for obtaining the NC membrane with different pore sizes and surface chemical properties includes: Add 0.3 - 0.9 g of PEG2000, 0.5 - 1.5 g of PEG6000, and 0.2 - 0.6 g of PEG20000 to the mixed solution respectively, and continue to stir at a stirring speed of 200 - 400 rpm for 2 - 4 h to obtain the second mixed solution; Slowly add 10 - 50 mL of 3 - aminopropyltriethoxysilane with a concentration of 0.5 - 1.5% to anhydrous ethanol, and stir at a stirring speed of 100 - 300 rpm at room temperature for 10 - 30 min to obtain the APTES solution; Immerse the pretreated first NC membrane in the APTES solution at 20 - 40°C for 1 - 6 h, and then take it out to obtain the second NC membrane; Take out the second NC membrane from the APTES solution and rinse it 3 - 5 times with anhydrous ethanol to obtain the third NC membrane; Immerse the third NC membrane in the PBS buffer solution at room temperature for 10 - 30 min, and then rinse it 2 - 3 times with deionized water to obtain the fourth NC membrane; Dry the fourth NC membrane in a vacuum oven at 40 - 60°C for 2 - 6 h; and cut the dried product to obtain the NC membrane with different pore sizes and surface chemical properties.

2. The preparation method of the fluorescence immunochromatographic detection card for lead and cadmium in tobacco according to claim 1, wherein, The preparation method of the MOFs powder includes: Dissolve the dried zinc nitrate in N,N-dimethylformamide, place it on a magnetic stirrer and stir until completely dissolved to prepare a first mixed solution with a concentration of 0.1-0.3 mol / L; Dissolve the recrystallized phthalic acid in N,N-dimethylformamide, place it on a magnetic stirrer and stir until completely dissolved to prepare a first composite solution with a concentration of 0.05-0.15 mol / L; Transfer the first mixed solution and the first composite solution to a stainless steel reaction kettle with a polytetrafluoroethylene liner according to a molar ratio of 1:1-2, and control the filling degree of the reaction kettle to be 60%-80%; seal the reaction kettle and place it in an oven at a temperature of 120-150 °C for reaction for 12-36 h to form MOFs crystals; Turn off the oven and wait for the reaction kettle to cool naturally to room temperature; filter the reaction solution in the reaction kettle through filter paper, collect the solid product on the filter paper, preliminarily separate the MOFs crystals and the reaction solvent, wash the collected solid product with DMF repeatedly for 3-5 times; then transfer the washed MOFs crystals to a Soxhlet extractor, use absolute ethanol as the extractant, and extract for 24-48 h; place the extracted MOFs crystals in a vacuum drying oven and dry at 80-100 °C for 10-20 h to obtain MOFs powder.

3. The preparation method of the fluorescence immunochromatographic test strip for lead and cadmium in tobacco according to claim 2, characterized in that, The method for preparing the labeled antigen includes: Step 1: Disperse the blank microspheres in a terbium chloride solution with a concentration of 0.01-0.1 mol / L at 20-60 °C, control the magnetic stirring speed at 100-500 rpm, after reacting for 12-48 h, centrifuge at 5000-10000 rpm for 5-15 min to discard the supernatant, obtain the microsphere precipitate, and wash the microsphere precipitate with an ethanol solution with a volume fraction of 70%-95% to obtain time-resolved fluorescence microspheres; Step 2: Activate the time-resolved fluorescence microspheres to obtain activated time-resolved fluorescence microspheres, and modify the activated time-resolved fluorescence microspheres based on polyethylene glycol derivatives to obtain modified time-resolved fluorescence microspheres; Step 3: Couple the lead antigen solution and the modified time-resolved fluorescence microspheres according to a molar ratio of 5-15:1 to obtain a first coupling solution; couple the cadmium antigen solution and the modified time-resolved fluorescence microspheres according to a molar ratio of 5-15:1 to obtain a second coupling solution; Step 4: Add BSA blocking solution to the first coupling solution and the second coupling solution respectively to block the vacant sites on the surface of the modified time-resolved fluorescence microspheres to obtain the first labeled antigen and the second labeled antigen; Step 5: Mix the first labeled antigen and the second labeled antigen according to a volume ratio of 1:1 to obtain the labeled antigen.

4. The preparation method of the fluorescence immuno-chromatographic detection card for lead and cadmium in tobacco according to claim 3, characterized in that, The method for obtaining the modified time-resolved fluorescence microspheres includes: Step 2.1: Suspend time-resolved fluorescent microspheres with a volume of 1 - 10 mL and a concentration of 5 mg / mL in a PBS buffer solution with a volume of 10 - 50 mL and a concentration of 0.01 - 0.1 mol / L. The PBS buffer solution contains EDC and NHS, and the concentrations of both EDC and NHS are 1 - 10 mM. Stir and react at a stirring speed of 200 - 400 rpm at room temperature for 30 - 60 min, then obtain a centrifugate through centrifugation at a speed of 5000 - 10000 rpm for 5 - 15 min, and remove the supernatant of the centrifugate. Wash the precipitate with the PBS buffer solution 2 - 3 times. After each washing, perform centrifugation at a speed of 5000 - 10000 rpm for 5 - 15 min to obtain the precipitate from the last centrifugation as the activated time-resolved fluorescent microspheres; Step 2.2: Resuspend the activated time-resolved fluorescent microspheres in a PBS buffer solution with a volume of 10 - 50 mL and a concentration of 0.01 - 0.1 mol / L. The PBS buffer solution contains NHS-PEG with a concentration of 1 - 10 mM, and stir at a stirring speed of 100 - 300 rpm at room temperature for 2 - 24 h to obtain a modification solution; Step 2.3: Perform precipitation treatment on the modification solution through centrifugation at 5000 - 10000 rpm for 5 - 15 min, remove the supernatant to obtain a precipitate, wash the precipitate 3 - 5 times with a PBS buffer solution with a volume of 10 - 50 mL and a concentration of 0.01 - 0.1 mol / L, and perform centrifugation at 5000 - 10000 rpm for 5 - 15 min after each washing to obtain the precipitate from the last centrifugation as the modified time-resolved fluorescent microspheres.

5. The preparation method of the fluorescence immunoassay chromatography test strip for lead and cadmium in tobacco according to claim 4, characterized in that, The method for obtaining the mixed solution includes: Slowly add 5 - 15 g of nitrocellulose with a purity greater than 95% and a molecular weight of 80000 - 150000 Da to a mixed solvent with a mass fraction of 10% - 15% and a volume of 50 - 150 mL, and stir and dissolve at a stirring speed of 200 - 400 rpm at room temperature until a uniform and transparent mixed solution is formed; wherein, the mixed solvent is a mixed solvent of acetone and DMF mixed in a volume ratio of 3 - 5:1; The method for obtaining the first NC membrane includes: Slowly drip 0.15 - 0.45 g of 3-aminopropyltriethoxysilane into the second mixed solution, and stir and react at a stirring speed of 200 - 400 rpm for 1 - 2 h to obtain a third mixed solution; Add 0.015 - 0.045 g of sodium dodecyl sulfate to the third mixed solution and stir for 30 - 60 min to obtain a formulated solution; Pour the formulated solution onto a clean glass plate placed horizontally, and use a scraper to uniformly scrape the film at a speed of 5 - 10 cm / s and a gap of 100 - 200 μm to obtain a liquid film; Immediately transfer the liquid film together with the glass plate to an environmental chamber with a temperature of 25 - 35°C and a humidity of 40% - 60% and place it for 12 - 24 h until the liquid film is completely dry; After the liquid film is dried, it is peeled off from the glass plate and rinsed 2 - 3 times with deionized water for 3 - 5 minutes each time to obtain a thin film; The thin film is immersed in absolute ethanol for 1 - 2 hours and then taken out and placed in a fume hood to dry; The dried thin film is dried in a vacuum oven at 40 - 50 °C for 4 - 6 hours to obtain the first NC film; The first NC film is immersed in absolute ethanol for 10 - 30 minutes to obtain the pretreated first NC film.

6. Detection method for a fluorescence immuno-chromatographic test strip for lead and cadmium in tobacco, characterized in that, Detect using the detection card prepared by the preparation method of the fluorescence immunoassay chromatography detection card for lead and cadmium in tobacco according to any one of claims 1 - 5, including the following steps: Take 5 - 10 g of tobacco samples, cut them into particles with a particle size of 1 - 3 mm, put them into a container containing 10 - 15 mL of aminotrimethylphosphonic acid with a concentration of 0.02 - 0.08 mol / L, and connect the container to the inlet of the micro - nano extraction device; Turn on the ultrasonic generating device, set the ultrasonic frequency to 20 - 50 kHz, the power to 100 - 300 W, and extract for 5 - 10 minutes; After the extraction is completed, use a hydrochloric acid solution with a concentration of 0.01 - 0.1 mol / L as the eluent to elute the lead and cadmium ions adsorbed on the extraction material, and collect the eluent as the sample to be detected for subsequent immunoassay; Drop 100 - 200 μL of the sample to be detected onto the sample well of the detection card prepared by the preparation method of the fluorescence immunoassay chromatography detection card for lead and cadmium in tobacco according to any one of claims 1 - 5, and incubate for 10 - 15 minutes; Insert the detection card into the supporting time - resolved fluorescence immunoassay analyzer, read the signal intensity values of the T - line and C - line, and process the signal intensity values to obtain the standard concentration of lead and cadmium in tobacco.

7. The detection method of the fluorescence immunochromatographic test strip for lead and cadmium in tobacco according to claim 6, wherein, The method for processing the signal intensity values to obtain the standard concentration of lead and cadmium in tobacco includes: Preset wavelength and , where is the emission wavelength of the labeled antigen; is the reference wavelength; Before inserting the test card and adding the sample to be tested, start the background monitoring program of the detection instrument; the instrument collects the fluorescence signal intensities at the positions of the T line and the C line at a preset frequency, continuously collects for 10 - 20 s, and obtains a set of background signal intensity data, denoted as , where , represents the emission wavelength of the selected antigen or the reference wavelength , represents the number of acquisitions; Calculate the average value of the background signal intensity at two wavelengths respectively and the standard deviation ; where is the total number of acquisitions, and take as the initial background subtraction value at wavelength and as the initial background subtraction value at wavelength , and set the confidence interval to , where is a constant 8. The detection method of the fluorescence immunochromatographic detection card for lead and cadmium in tobacco according to claim 7, characterized in that, Drop the extraction solution of a pre-prepared standard sample with known lead and cadmium concentrations onto the test card, insert it into a time-resolved fluorescence immunoassay analyzer, and obtain the real-time signal intensity values of the T line and the C line at wavelengths and , denoted as , , and , where is the real-time signal intensity of the T line at wavelength ; is the real-time signal intensity of the T line at wavelength ; is the real-time signal intensity of the C line at wavelength ; is the real-time signal intensity of the C line at wavelength ; is the th standard sample; simultaneously collect the background signal intensity at this time; Calculate the signal intensity ratio of the T line at wavelengths and , and the signal intensity ratio of the C line at wavelengths and ; for each standard sample, calculate its corresponding lead-cadmium concentration ratio; Using the least squares method in combination with the signal intensity ratio, fitting and establishing the T-line correction coefficient and the C-line correction coefficient and the linear relationship with the concentration ratio ; respectively calculating and obtaining the coefficients and intercepts corresponding to the T-line and the C-line.

9. The detection method of the fluorescence immunochromatographic detection card for lead and cadmium in tobacco according to claim 8, characterized in that Collect the total signal intensities of the T-line and C-line at two wavelengths at preset time intervals , , and , where is the total signal intensity of the T-line at wavelength ; is the total signal intensity of the T-line at wavelength ; is the total signal intensity of the C-line at wavelength ; is the total signal intensity of the C-line at wavelength ; At the same time, collect the background signal intensity , where , corresponding to the background signal intensity at wavelength and the background signal intensity at wavelength respectively; If is within the confidence interval, then use as the background subtraction value, and calculate the net signal intensity by subtracting the background subtraction value from the total signal intensity; If it exceeds the confidence interval, the background average value obtained by recollecting the background signal and calculating is used as the new background subtraction value to update the net signal intensity; Calculate the effective signal strength of the T line and the effective signal strength of the C line after compensation; the effective signal strength is obtained by calculating the difference between the net signal strength of the T line at wavelength and the result of multiplying the net signal strength of the T line at wavelength by the adjustment coefficient, where the adjustment coefficient is the ratio of the product of the net signal strength of the T line at wavelength and the coefficient to the product of the coefficient, the estimated ratio of the lead and cadmium concentrations in the real-time detection sample, and the intercept; Using the standard curve established in advance through standard samples, combined with the effective signal intensity of the compensated T - line, calculate the concentration of lead and cadmium in the sample to be detected by interpolation method or regression equation.

10. The detection method of the fluorescence immunoassay chromatographic test strip for lead and cadmium in tobacco according to claim 9, characterized in that, The method for calculating the concentration of lead and cadmium in the sample to be detected by interpolation method or regression equation includes: The effective signal intensity of the T line after compensation on the calibration curve of two adjacent data point coordinates and , where the effective signal intensity of the T line after compensation is located and ; the concentration of lead and cadmium in the sample to be measured is calculated by multiplying the ratio of the difference in the abscissas of two adjacent data points to the difference in the ordinates, multiplying by the difference between the effective signal intensity and the ordinate of one of the data point coordinates, and then adding the abscissa of the corresponding data point coordinate.

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